Blood samples (20 ml) were collected from 12 patients (Patient number P1-P12) who were diagnosed with cervical cancer at Cancer Institute Adyar, Chennai for isolation of PBMC and DC culture. Tumour punch biopsy samples were collected from 3 patients (Patient number P1-P3) to prepare tumour lysates for the generation of TLDCs. In addition, 20ml blood samples were also obtained from eight healthy donors for isolation of PBMCs using Ficoll (GE Healthcare, US). The monocytes were depleted by plastic adhesion subsequently to enrich for lymphocytes and used for allogeneic studies. The study was approved by the Institutional ethical committee from Cancer Institute Adyar, Chennai. The duly signed consent forms were obtained from each patient and healthy donor prior to the study. Tumour tissues were collected for tumour lysate in Hank’s balanced salt solution (HBSS) containing 100IU of penicillin, streptomycin and gentamycin and in 10% formalin for immunohistochemistry.
Immunohistochemistry for SPAG9 expression
The SPAG9 protein expression was examined in cervical cancer tissue sections by IHC as described earlier . Briefly, the cervical cancer tissue sections (4μm) were deparaffinized and subsequently rehydrated using different gradients of alcohol. Polyclonal antibodies to recombinant SPAG9 were generated and purified as described earlier . Tissue sections were probed with anti-SPAG9 antibody or control IgG at 1:100 dilution overnight at 4°C in humid chamber. After washing thrice with phosphate buffer saline (PBS)-0.05% Tween 20, the tissue sections were incubated with horseradish peroxidase-conjugated goat anti-rat IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) and visualized using DAKO Envision kit (K500711-2) following the manufacturer’s instructions and counterstained with hematoxylin and mounted with 1,3-diethyl-8-phenylxanthine [(DPX), Sigma-Aldrich, St. Louis, MO]. The images of tissue sections were captured using Nikon microscope (Nikon Instrument lnc., NY, USA).
Antibodies and flow cytometry
For phenotypic analysis of dendritic cells: Fluorochrome conjugated antibodies CD14-PC5 (CD-cluster of differentiation PC5- PE- Phycoerythrin cy5-cyanine, CD80-FITC, HLADR-ECD, CD40-PE and CD86-PE were purchased from Beckman Coulter Inc (CA, USA). The antibody against maturation marker CD83-conjugated to APC was purchased from Bio Legend (San Diego, CA, USA)
For phenotyping proliferating PBMCs, anti- CD56-PE, anti-CD4-PC5 anti-CD8-APC, anti- CD25-ECD and anti-CD3-PC7 antibodies were purchased from Beckman Coulter Inc. Anti-FOXP3-PE was purchased from BioLegend Inc. Flow cytometry was performed as described previously . All the samples were acquired using MoFlo XDP flow cytometer configured with three different lasers - blue (488nm), violet (405nm) red (640nm) analyzed using Summit 5.2 software (both Beckman Coulter Inc).
DC generation and priming
Unprimed and TLDCs were generated from peripheral blood mononuclear cells (PBMCs) isolated using a Ficoll Paque Plus (GE Healthcare, UK) density gradient. Monocytes were enriched by plastic adherence for DC differentiation and were either primed with tumour lysates (TLDCs) prepared from single cell suspensions of punch biopsy samples or matured directly (unprimed DCs) as described earlier . SPDCs were generated using GMP grade rhSPAG9 (GMP grade rhSPAG9 was outsourced for manufacturing to Syngene International Pvt. Ltd, India) . The purified rhSPAG9 protein was used at different concentrations such as 250 ng, 500 ng, 750 ng and 1000 ng/ml for priming and generation of SPDCs following same protocol used for the generation of TLDCs.
Wash out test
The mature dendritic cells were washed and suspended in serum free CellGro DC medium (Cell Genix, Freiburg, Germany) for 24h. Adherence and changes in morphology following cytokine withdrawal were monitored microscopically and counts were obtained by trypan blue exclusion. The washout test medium was stored in protein low bind tubes (Eppendorf, Hamburg, Germany) at -70oC.
Phenotypic characterization of mature DCs
For immunophenotypic analysis, 5x104 mature DCs were incubated for 10 min with 5% FBS and then stained with anti- CD14 PC5, CD40 PE, HLADR ECD, CD86 PE, CD80 FITC and anti CD83 APC for 20 min at RT. Cells were also stained with appropriate Isotypic controls and washed twice with PBS, fixed and suspended in 2% PFA (Sigma Aldrich, MI, USA) until analysis.
Proliferation assay with rhSPAG9 primed DCs
For the proliferation assay, non adherent cells enriched for lymphocytes after monocyte depletion for DC culture were prepared from allogenic and autologous PBMCs. Matured DCs primed with rhSPAG9 (250, 500, 750 and 1000 ng/ml per million cells), were cocultured with PBMCs stained with carboxy fluorescein, succinimidyl ester [CFSE (Invitrogen, CA, USA)]. Briefly, SPDCs were cocultured at the ratio of 1:50 (DC: allogeneic PBMCs) and 1:10 (DC: autologous PBMCs) to check for proliferation as described in our previous study .The cells were cultured for 8 days. The cells were centrifuged at 1580 rpm and subsequently pellet was resuspended and washed with PBS, blocked with 5% FBS and stained with anti-CD4-PC5, anti-CD8-APC, anti-CD56-PE, anti-CD25-ECD, anti-FOXP3-PE antibodies and propidium iodide (1µg/ml; Himedia, India) to verify the response of the autologous or allogeneic PBMCs upon stimulation with SPDCs. Cells were acquired using a MoFlo XDP cell sorter/ Flow cytometer (Beckman Coulter) and FCS express 7 was used for analyzing the proliferating population.
Migratory capacity of TLDCs and SPDCs was assessed in a 24 well plate using (5µ) transwell inserts (Corning, NY, USA). The lower chamber of each well was filled with serum free medium containing 300ng/ml CCL19 and 250ng/ml CCL21 (R&D systems, MN, USA) and the DCs were incubated for 3h at 37°C. Subsequently, the cells were centrifuged and then re-suspended in total volume of 300µl PBS. Samples were analyzed in a flow cytometer using a fixed flow rate. The number of cells acquired per minute through flow cytometer was calculated as described previously .
IFNγ Analysis by ELISPOT assay
ELISPOT assay was done to measure antigen-specific IFNγ release by PBMCs removed after monocytes attachment. All steps were carried out as per manufacturer’s protocol [CTL Technologies, OH, USA]. The matured DCs (TLDCs or SPDCs or unprimed DCs) were incubated with allogenic non adherent PBMCs at a 1:50 ratio for 24h at 37°C /5% CO2 in IFNγ captured antibody coated wells. The lymphocyte enriched autologous PBMCs were also cultured similarly with matured DCs at 1:10 ratio for 2 weeks and incubated for 48h in IFNγ captured antibody coated wells in the presence of 100 IU/ml IL2 along with respective matured DCs. To avoid inter sample variation we multiplied the spot count with the spot size which is represented as the IFNγ spot Index for each patient. Following manufacturer's instructions spots were scanned using Immunospot versa analyser (CTL, OH, USA) using ImmunoCapture software version 6.4. The spots were then counted using the ImmunoSpot 5.0 ProDC software.
Cytokines Analysis by ELISA
Patients (patient number P7-P9) monocytes derived DC either primed with rhSPAG9 or tumour lysates were generated in 7 days. Post 7 days, SPDCs and TLDCs were cultured in plain Cell Gro medium for 24h without the proinflammatory cytokine. All viable floating matured DCs were collected for DCs-PBMCs cocultures experiments and supernatant (WO) was stored at −80°C for conducting the ELISA experiments. Subsequently, autologous DCs thus generated were further co-cultured with PBMCs for 7 days. Supernatants from each well were collected on Day 7 and stored at −80°C for conducting ELISA experiments. Both IL12p70 and p40 were quantitated separately using the respective ELISA kits (IL12p70 and IL12p40 kit; Biolegend, CA, USA) according to the manufacturer's instructions. The color developed was read at 450nm using Multiskan Ascent (Thermo Electron Corporation, USA).
Effect of Cisplatin treatment on autologous PBMC cocultures with dendritic cells
To examine the effect of Cisplatin (Kemoplat, Fresenius Kabi, Germany) on DC function, three different concentrations -150µM (Dose 1), 200µM (Dose 2) and 400µM (Dose 3) (where Dose 2 is equivalent to the clinical dosage) were used. SPDCs were seeded (1X105 cells) into 96 well plate and cultured for 24h at 370C followed by three different doses of cisplatin treatment. Treated SPDC were cocultured with autologous PBMC in 1:10 ratio (n=3; patient number P10-12). The frequency of treatment was day 0, 3 and 5 for monitoring CFSE based proliferation, for up to eight generations which is usually attained within a week. DCs alone as control were treated for 48h with cisplatin and phenotypic marker expression and migratory capacity were analyzed. All experiments were carried out in three independent patients (P10-P12).
Mann-Whitney U test was done to compare unprimed DCs with SPDCs and TLDC with SPDCs. One-way ANOVA (Analysis of variance) was used to compare the phenotypic and functional characteristics of untreated and cultures treated with three different doses of CDDP. All the analyses were done using GraphPad prism 7 software (GraphPad Software, Inc. USA).